Influence of Cool Storage before Freezing on the Quality of Frozen-Thawed Semen Samples in Dogs
Unit of Reproduction and Obstetrics, Veterinary Faculty, University of Las Palmas de Gran Canaria, Arucas, Las Palmas, Spain.Reproduction in Domestic Animals (Impact Factor: 1.52). 06/2012; 48(1). DOI: 10.1111/j.1439-0531.2012.02124.x
The aim of this study was to determinate the semen quality of frozen-thawed samples that were chilled for up to 2 days before freezing. The ejaculates (n = 18) from six dogs were collected, pooled and divided into six aliquots. The first aliquot (C, control) was frozen in liquid nitrogen using a conventional protocol to reach a final concentration of 100 × 10(6) spermatozoa/ml, 20% egg yolk and 5% glycerol. The remaining five aliquots were diluted with a chilled extender (Tris-glucose and 20% egg yolk) and cooled at 4°C as follows: R1, the semen was cooled for 1 h; R6, the semen was cooled for 6 h; R12, the semen was cooled for 12 h; R24, the semen was cooled for 24 h and R48, the semen was cooled for 48 h. After the chilling period, a second extender was added (Tris-glucose, 20% egg yolk, 10% glycerol and Equex at 1%) to reach a final composition similar to aliquot C, and then, the semen samples (R1, R6, R12, R24 and R48) were frozen in liquid nitrogen. The post-thaw sperm quality was assessed in 30 straws from each experimental group. After freezing-thawing, the total sperm motility (approximately 60-70%) in the semen chilled for up to 48 h did not show any differences from the samples frozen by the conventional cryopreservation method (63.2%). No significant differences were detected in the percentages of abnormal sperm cells among the fresh semen, the control group and the frozen samples after the different cooling times. Finally, the post-thaw percentages of damaged acrosomes showed a very uniform distribution, with mean values ranging between 7% and 10.5%. The results clearly demonstrated that cooling the semen up to 48 h before freezing did not produce a decrease in the semen quality when was compared with semen frozen by a traditional procedure.
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ABSTRACT: This study aimed to assess the influence of cool storage (5 °C) prior cryopreservation over the post-thaw quality of buck semen samples. Semen of six Majorera bucks (n = 18 ejaculates) was collected, pooled and diluted in a Tris-yolk extender. Then, diluted semen was divided into six aliquots; the first aliquot (group C) was processed and frozen in liquid nitrogen (final concentration of 400 × 106 spermatozoa/mL, 12% egg-yolk and 4% glycerol). The remaining aliquots (diluted with Tris-glucose, 12% egg yolk) were hold for 1 to 48 hours at 4 °C: R1, the semen was cooled for 1 hour; R6, the semen was cooled for 6 hours; R12, the semen was cooled for 12 hours; R24, the semen was cooled for 24 hours and R48, the semen was cooled for 48 hours. After each cooling period, a second extender was added to reach a final composition (400 × 106 spermatozoa/mL, 12% egg-yolk and 4% glycerol) similar to group C; finally, semen was packed and frozen in liquid nitrogen. After freezing-thawing, the sperm motility, acrosome integrity and the percentage of abnormal spermatozoa were assessed. No differences (P > 0.05) were detected in progressive sperm motility (mean range: 35.4-39.9%) and damage acrosomes percentages (mean:10.8-15.5%) among the control group and the cooled semen samples (R1, R6, R12, R24) for up to 24 hours; however, R48 samples showed a lower (21.6%, P < 0.01) progressive fast spermatozoa percentage and a higher percentage of damage acrosome (38.3%, p < 0.01) than those observed in the control group and in R1, R6, R12 and R24 samples. The present study confirmed that buck semen could be preserved at 5 °C for up to 24 hours before freezing; however, after 2 days of chilling, semen quality experienced a notable decrease and its utility could be lower.
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ABSTRACT: The aim of this work was to assess the combined effect of sperm centrifugation, semen extender and storage time before freezing on post-thaw sperm quality and freezability on chilled stored canine semen in a Neopor box. Sperm parameters evaluated were total and progressive sperm motility by Computer-Assisted Sperm Analysis (CASA) and sperm viability and acrosome integrity using a triple fluorescent stain. Sperm quality and freezability indexes were also studied. First, the effect of centrifugation and two commercial extenders from Minitübe (Biladyl A and CaniPRO Freeze A) was evaluated in chilled semen after 24 and 45 hours of cold storage. No significant differences were observed between treatments in almost all the sperm parameters assessed. Secondly, chilled semen was frozen after 24 and 45 hours of cold storage in a Neopor box. The best results were obtained when semen was centrifuged, chilled with CaniPRO Freeze A and then frozen after 24 hours of cold storage, showing no differences in both post-thaw sperm quality and freezability in comparison with semen immediately frozen after collection. In conclusion, dog semen centrifuged after collection and extended with CaniPRO Freeze can be frozen after 24 hours of cold storage in a Neopor box, obtaining similar results to semen immediately frozen after collection.
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ABSTRACT: The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.
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